ELISA Enzyme Substrates

Figure 1. Options for incorporating detection enzymes like HRP or AP into ELISA. In the assay, the antigen of interest is immobilized by direct adsorption to the assay plate or by first attaching a capture antibody to the plate surface. Detection of the antigen can then be performed using an enzyme-conjugated primary antibody (direct detection) or a matched set of unlabeled primary and conjugated secondary antibodies or biotin-labeled primary antibody detected using enzyme-linked streptavidin (indirect detection).

ELISA substrates are also available as OEM or Commercial Supply versions. Inquire about reagents and consumables, instruments, and custom kitting package solutions.

ELISA Builder can make it easy to find a great solution for developing your own ELISA assay. Based on a short list of questions about your experimental needs, the ELISA Builder tool recommends everything needed to perform an ELISA, from plates to stop solution.

TMB (3,3',5,5'-tetramethylbenzidine) is a soluble substrate that yields a blue color when detecting HRP. It is very sensitive and may produce significant background signal if too much protein or antibody is used. TMB is more quickly oxidized than other HRP enzyme substrates, resulting in faster color development.

We have different formulations to choose from, depending on your experimental design and sensitivity needs.

See Figure 2 for a comparison of the range of sensitivities for the different formulations and selection guide (Tab 1) for the optimal kit for your experiment.

ABTS (2,2'-Azinobis [3-ethylbenzothiazoline-6-sulfonic acid]-diammonium salt) is used to detect HRP and yields a water-soluble green end reaction product.

ABTS is less sensitive than the OPD and TMB substrates for HRP detection. Color development is slow (approximately 20 minutes) which may be advantageous if unacceptable background results from the use of the OPD or TMB substrates due to higher sensitivities.

ABTS is available in either tablet or a ready-to-use for formulation.

OPD (o-phenylenediamine dihydrochloride) is used to detect HRP and yields a water soluble yellow-orange reaction product. The reaction product has an absorbance maximum of 492 nm.

OPD is available in either powder or tablet forms and easily prepared by dissolving in Stable Peroxide Substrate Buffer or buffered hydrogen peroxide solution.

Figure 2. Sensitivity comparison of various TMB colorimetric ELISA substrates for HRP. TMB (3, 3’, 5, 5’-tetramethylbenzidine), a common chromogenic substrate for HRP, yields a blue color when oxidized. The color then changes to yellow with the addition of sulfuric or phosphoric acid, common solutions used to stop the reaction. In graph on the left, the performance of multiple TMB substrates is compared in an ELISA plate assay.

SuperSignal ELISA Pico Chemiluminescent Substrate provides excellent performance for a large range of target protein amounts and is easily optimized to detect with greater sensitivity than entry-level colorimetric substrates. Rapid signal generation with 5–30 minutes signal stability depending on HRP concentration (Figure 3).

SuperSignal ELISA Femto Maximum Sensitivity Substrate is one of the most sensitive substrates available for ELISA applications. When properly optimized, the lower detection limit is 1 to 10 orders of magnitude lower than commonly used colorimetric substrates. However, without proper optimization, it is easy to overwhelm the system with protein and enzyme, resulting in high background and possibly negative results.

Figure 3. Immediate light generation with SuperSignal ELISA Pico Chemiluminescent Substrate. Biotinylated HRP or biotinylated AP (200 pg) were added to separate wells of NeutrAvidin-coated white polystyrene plates. The plates were then incubated for 30 minutes at room temperature (RT) on a plate shaker and then each well was washed three times with BupH Tris-Buffered Saline (Cat. No. 28379). Working solutions of chemiluminescent substrates were prepared according to the manufacturers’ instructions. SuperSignal ELISA Pico Chemiluminescent Substrate (Cat. No. 37070) and another luminol-based system (Brand A), 100 µL of each substrate working solution was added to the appropriate plate well. For the dioxetane-based system, wells were washed with 1X Assay Buffer. Next, 100 µL of the dioxetane working solution was added to the appropriate plate well. All plates were incubated on a plate shaker at RT for 1 minute. Plates were then read on a Dynex MLX Microtiter Plate Luminometer with a 0.2 second read time per well. Several readings were taken over a 30-minute period.

QuantaBlu Fluorogenic Substrate has a large linear detection range with low-end linearity for detection of HRP. The stable fluorescent reaction product has an Ex/Em of 325 nm/420 nm allowing stopped, non-stopped, and kinetic assays to be performed; an advantage over the more sensitive chemiluminescent substrates.

QuantaRed Enhanced Chemifluorescent Substrate is the most sensitive fluorescent ELISA substrate available for HRP detection. The fluorescent reaction product (resorufin) is stable for 4 hours with an Ex/Em of 570 nm/585 nm when the reaction is stopped. The red-shifted resorufin reaction product permits detection at a wavelength with less interference from autofluorescence that can occur in biological samples.

Amplex Red Reagent (10-acetyl-3,7-dihydroxyphenoxazine) in the presence of horseradish peroxidase reacts with hydrogen peroxide in a 1:1 stoichiometry to produce highly fluorescent resorufin. This HRP enzyme substrate has greater stability, yields less background and produces a red-fluorescent product (Ex/Em of 571/585 nm) that is detected via colorimetric absorbance or fluorescence emission.

Amplex UltraRed Reagent improves upon the performance of Amplex Red reagent, offering brighter fluorescence and enhanced sensitivity on a per-mole basis in horseradish peroxidase or horseradish peroxidase–coupled enzyme assays. Fluorescence of oxidized Amplex UltraRed reagent is also less sensitive to pH, and the substrate and its oxidation product exhibit greater stability than Amplex Red reagent in the presence of H2O2 or thiols such as dithiothreitol (DTT).

The Amplex Red/UltraRed Stop Reagent (Cat. No. A33855) provides convenience and control by allowing the fluorescence signal-generating reaction to be terminated at a user-determined time point.

PNPP (p-Nitrophenyl Phosphate, Disodium Salt) is a widely used substrate for detecting alkaline phosphatase in ELISA applications. PNPP produces a yellow water-soluble reaction product that absorbs light at 405 nm. PNPP is available either as a crystalline powder, 5 mg tablets, or as a ready-to-use formulation.

ONPG (o-nitrophenyl-β-D-galactopyranoside) is used in ELISA applications involving beta-galactosidase (β-Gal). ONPG produces a completely soluble yellow product with a high extinction coefficient at 405 nm. While other β-Gal substrates exist, ONPG’s high solubility, ease of workflow, and significant signal-to-noise ratio make it the most ideal choice.

CSPD and CDP-Star substrates for AP

CSPD and CDP-Star are chemiluminescent 1,2-dioxetane alkaline phosphatase substrates that emit light with a maximum light intensity at a wavelength of 475 nm. These substrates are “glow” substrates and provide a sustained maximum signal over time only after 15–60 minutes depending on temperature. They are supplied at 5 or 25 mM (respectively) in aqueous buffer. Sensitivity can be improved with addition of Sapphire-II or Emerald-II luminescence enhancers.

CDP-Star substrate produces a higher signal in solution assays. CDP-Star substrate with either Sapphire-II or Emerald-II enhancer produces a signal that is nearly five-fold higher than the signal produced by CSPD substrate with enhancer. For five-fold maximum sensitivity, CDP-Star Substrate solutions will provide the highest signal intensity and the greatest sensitivity.

DynaLight Substrate for AP

DynaLight Substrate with RapidGlow Enhancer is a ready-to-use chemiluminescent substrate formulation that has been optimized to achieve faster results in solution-based assays. This substrate is classified as a flash and glow substrate that provides fast and sustained maximum signal as early as 2–10 minutes. The DynaLight Substrate with RapidGlow Enhancer formulation includes 1,2-dioxetane chemiluminescent substrate and a polymeric enhancer that enables ultra-sensitive immunoassay detection by alkaline phosphatase label.

Figure 4. Signal kinetics comparison of Glow and Flash and Glow substrates. Comparing signal intensity kinetics (panel A) and signal-to-noise ratios (panel B) of “Glow” and “Flash and Glow” substrate formulations. 0.04 ng of biotin-alkaline phosphatase was detected using 20 µg of M-280 SA Dynabeads magnetic beads in a white microplate. Signal kinetics at 25°C are shown for both “Glow” (i.e., CDP-Star Substrate with Emerald-II Enhancer) and “Flash and Glow” (i.e., DynaLight Substrate with RapidGlow Enhancer) substrates, respectively reaching maximum light emission at ~30 minutes and ~5–10 minutes.

ELISA Kits and Components

Learn about our ELISA kit formats including complete, ready-to-use kits as well as preoptimized reagents to design your own assay.

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ELISA Blocking Buffers and Reagents

Select from a wide variety of easy-to-use and reliable blocking buffers and reagents for ELISA applications.

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ELISA Instrumentation and Equipment

We offer centrifuges, cold storage equipment, dispensers, incubator/shakers, microplates, pipettes and tips, and microplate readers and washers to help meet all your ELISA needs.

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ELISA Microplates and Plasticware

Choose from an array of microplates with a variety of surfaces optimized to capture the biomolecule of your choice.

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